The project?s long term goal is to define the mechanisms used by neutrophils (PMN) to regulate epithelial barrier function. The tight junction (TJ) acts as a regulated barrier for diffusion of ions, larger solutes, and migrating cells through the paracellular space. Altered Ti structure and function have been correlated with enhanced migration of PMN through the intestinal crypt epithelium in idiopathic inflammatory bowel disease (IBD). Our central hypothesis is that PMN applied to the basolateral aspect of epithelial monolayers enhance epithelial paracellular permeability in a polarized manner independent of transmigration, but requires cell-cell adhesion and a chemotactic gradient. We will first characterize PMN-stimulated functional changes to epithelial barrier function using an established in vitro model system of epithelial monolayers and human PMN. Second, under this process at the molecular level, PMN-stimulated monolayers will be analyzed by microscopic localization of components of TJ and the actin-cytoskeleton. Alterations in the localization of key structural and signaling components of the TJ will be studied using biochemical methods, including changes in TJ protein composition, phosphorylation and protein-protein interactions. This work will yield insight into mechanisms used by PMN to alter epithelial barrier function and provide new ideas for therapeutic intervention in IBD.